A double qPCR method for detecting psittacine beak and feather disease virus and avian polyomavirus, and primers and probes

Abstract

The application discloses a double qPCR method, primers and probes for detecting psittacine beak and feather disease virus and avian polyomavirus, which comprises a first primer pair and a first probe for detecting the psittacine beak and feather disease virus, wherein the nucleotide sequence of the first forward primer is shown as SEQ ID NO: 3, the nucleotide sequence of the first reverse primer is shown as SEQ ID NO: 4, and the nucleotide sequence of the first probe is shown as SEQ ID NO: 5; and a second primer pair and a second probe for detecting the avian polyomavirus, wherein the nucleotide sequence of the second forward primer is shown as SEQ ID NO: 8, the nucleotide sequence of the second reverse primer is shown as SEQ ID NO: 9, and the nucleotide sequence of the second probe is shown as SEQ ID NO: 10. The application adopts double fluorescent quantitative PCR technology, can simultaneously detect the avian polyomavirus and the psittacine beak and feather disease virus in the same reaction tube, has no cross reaction with non-target pathogens, significantly saves time and reagent cost, and has high detection flux.

Description

Technical Field

[0001] This invention belongs to the field of PCR detection, specifically relating to a dual qPCR method, primers, and probes for detecting parrot beak feather disease virus and avian polyomavirus. Background Technology

[0002] Psittacine beak and feather disease (PBFD) is a serious infectious disease caused by the psittacine beak and feather disease virus (PBFDV). PBFDV is an immunosuppressive pathogen that, upon infection, attacks core immune organs such as the bursa of Fabricius and thymus, leading to immunosuppression. Although infected parrots do not die immediately, their impaired immune function makes them highly susceptible to secondary bacterial, fungal, or other viral infections, ultimately resulting in death from mixed infections. Avian polyomavirus (APV) is another pathogen that seriously harms parrots and other birds. APV has a wide host range, infecting various domestic and wild birds such as budgerigars, pigeons, and munias, with parrots being the most susceptible, and severe cases can lead to death.

[0003] In clinical practice, APV infection often occurs in combination with PBFDV. Parrots infected with PBFDV are highly susceptible to secondary infections with other pathogens such as APV due to immunosuppression. Co-infection of both can lead to a mortality rate of over 80% in chicks. Epidemiological surveys show that PBFDV and APV are prevalent in both captive and wild bird populations, with co-infection being more common in captive parrots. Detection methods for PBFDV and APV mainly include conventional PCR and real-time quantitative PCR. Existing technologies have reported detection of single viruses, such as using PCR technology to detect PBFDV or APV separately. A dual real-time quantitative PCR method based on TaqMan probes has been established to detect the PBFDV V1 gene and the APV T gene. Furthermore, Chinese invention patent application CN202410318389.8 also discloses a real-time quantitative PCR primer and probe combination, kit, and method for simultaneously detecting PBFDV and APV.

[0004] However, the existing technologies mentioned above still have certain limitations. On the one hand, the target genes and primer / probe sequences used in different studies are different, and the detection sensitivity also varies. The detection performance of different methods needs to be further improved and optimized. On the other hand, the target gene regions used in the existing methods are different, and there are no reports on dual TaqMan probe fluorescence quantitative PCR detection methods for PBFDV Rep gene and APV VP1 gene.

[0005] Therefore, it is necessary to establish a dual qPCR method, primers, and probes for detecting parrot beak feather disease virus and avian polyomavirus to meet the urgent needs of clinical testing and epidemiological monitoring in the parrot farming industry. Summary of the Invention

[0006] The purpose of this invention is to provide a dual qPCR method, primers, and probes for detecting parrot beak feather disease virus and avian polyomavirus.

[0007] This invention is achieved through the following technical solution: The present invention includes a first primer pair and a first probe for detecting parrot beak feather disease virus, comprising a first forward primer and a first reverse primer, wherein the nucleotide sequence of the first forward primer is shown in SEQ ID NO:3, the nucleotide sequence of the first reverse primer is shown in SEQ ID NO:4, and the nucleotide sequence of the first probe is shown in SEQ ID NO:5. The second primer pair and the second probe for detecting avian polyomavirus comprise the first forward primer and the second reverse primer, wherein the nucleotide sequence of the second forward primer is shown in SEQ ID NO:8, the nucleotide sequence of the second reverse primer is shown in SEQ ID NO:9, and the nucleotide sequence of the second probe is shown in SEQ ID NO:10.

[0008] Furthermore, the first probe has a VIC fluorescent group labeled at its 5' end and a BHQ1 quenching group labeled at its 3' end; the second probe has a FAM fluorescent group labeled at its 5' end and a BHQ1 quenching group labeled at its 3' end.

[0009] Furthermore, a dual real-time PCR kit for the simultaneous detection of parrot beak feather disease virus and avian polyomavirus comprises the aforementioned primer and probe combination.

[0010] Furthermore, it also includes a positive plasmid standard containing the Rep gene fragment of psittacosis virus and the VP1 gene fragment of avian polyomavirus. The positive plasmid standard is cloned and constructed by amplifying the Rep gene fragment of psittacosis virus and the VP1 gene fragment of avian polyomavirus using the first construction primer pair SEQ ID NO:1 and NO:2 and the second construction primer pair SEQ ID NO:6 and NO:7, respectively.

[0011] Furthermore, a dual-fluorescence quantitative PCR method for simultaneously detecting psittactic beak feather disease virus and avian polyomavirus, using the aforementioned primer and probe combination for dual-fluorescence quantitative PCR amplification, includes the following steps: (1) Extract total nucleic acid from the sample to be tested, and use the total nucleic acid as a template. (2) Collect fluorescence signals and determine the presence of parrot beak feather disease virus and / or avian polyomavirus in the sample based on the amplification curve and Ct value.

[0012] Further, the following are the contents of the 2×TaqMan probe-based real-time PCR premix: 10 μL of premixed solution, 0.2-0.4 μL each of the second forward primer and the second reverse primer, 0.2-0.4 μL each of the first forward primer and the first reverse primer, 0.6-0.8 μL each of the second probe and the first probe, 2 μL of DNA template, and the remainder being nuclease-free water, for a total volume of 20 μL.

[0013] Furthermore, the reaction procedure for the dual fluorescence quantitative PCR is as follows: 95°C pre-denaturation for 30 seconds; 40 cycles of 95°C denaturation for 5 seconds, 58°C annealing and extension for 34 seconds.

[0014] Furthermore, the sample to be tested is selected from liver tissue, cloacal swabs, or feather swabs.

[0015] The most significant feature of this invention compared to existing technologies is that the above-described technical solution is: This invention significantly reduces the risk of nonspecific amplification by employing specific primer and probe combinations targeting the bpe275 and bab1_1216 genes, ensuring the accuracy and reliability of detection results. It utilizes dual fluorescence real-time quantitative PCR technology to complete dual-target detection in a single reaction, significantly improving detection efficiency and making it suitable for large-scale sample testing. Attached Figure Description

[0016] Figure 1 PCR electrophoresis diagrams of the VP1 gene (A) of APV and the Rep gene (B) of PBFDV; Figure 2 Results of optimization of dual real-time quantitative PCR reaction conditions; Where A represents APV primer concentration optimization; B represents APV probe concentration optimization; C represents APV annealing temperature optimization; D represents PBFDV primer concentration optimization; E represents PBFDV probe concentration optimization; and F represents PBFDV annealing temperature optimization.

[0017] Figure 3 Standard curves for APV and PBFDV; Where A is the standard curve of APV; B is the standard curve of PBFDV.

[0018] Figure 4 Specific detection results of dual real-time quantitative PCR; Figure 5 Amplification curves of standard plasmids at different concentrations; Wherein, A is the amplification curve of pEASY-APV; B is the amplification curve of pEASY-BFDV, and the final concentration range of the plasmid is 1×10⁻⁶. 9 Up to 1×10⁻¹ copies / μL.

[0019] Figure 6 PROBIT analysis results of the detection limit of dual qPCR; Where A is the PROBIT analysis of pEASY-APV; B is the PROBIT analysis of pEASY-BFDV. Detailed Implementation

[0020] The technical solution of the present invention will be further described below with reference to embodiments and comparative examples, but it should not be construed as a limitation of the present invention: The objective of this invention is achieved through the following technical solution: Example 1: Design and Synthesis of Primers and Probes This embodiment provides a method for designing primer and probe combinations for the simultaneous detection of parrot beak feather disease virus (PBFDV) and avian polyomavirus (APV). The gene sequences of APV and PBFDV were downloaded from the GenBank database, and multiple sequence alignment was performed using MEGA software. Specific primers and TaqMan probes were designed within conserved regions of the VP1 gene of APV and the Rep gene of PBFDV, respectively.

[0021] The first primer pair and first probe sequence designed for the PBFDV Rep gene are as follows: First forward primer (BFDV-F1): 5'-tacttacyctsggcattgtggcga-3' (SEQ ID NO:3); First reverse primer (BFDV-R1): 5'-tgacgtagatatcHgggaact-3' (SEQ ID NO:4); The first probe (BFDV-P) is 5'-agagacggtcaccgcgctttcgat-3' (SEQ ID NO:5). The 5' end of this probe is labeled with a VIC fluorescent group, and the 3' end is labeled with a BHQ1 quenching group.

[0022] The second primer pair and second probe sequences designed for the APV VP1 gene are as follows: Second forward primer (APV-VP-F1): 5'-ccacagcacagaggtaccgtgt-3' (SEQ ID NO:8); Second reverse primer (APV-VP-R1): 5'-gtatccgttaaatgcgcgga-3' (SEQ ID NO:9); The second probe (APV-P) is 5'-tacagctgtgccaggataccgct-3' (SEQ ID NO:10). The 5' end of this probe is labeled with a FAM fluorescent group, and the 3' end is labeled with a BHQ1 quenching group.

[0023] In addition, this embodiment also provides long-fragment amplification primers for constructing positive plasmid standards: BFDV-F: 5'-atgccgtccaaggagggctctggct-3' (SEQ ID NO: 1); BFDV-R: 5'-gcgaggaaatcaggccggacttgt-3' (SEQ ID NO: 2); APV-VP-F: 5'-ggaagctgcccacgacctcagca-3' (SEQ ID NO: 6); APV-VP-R: 5'-gagtttagcaaaccagatacagga-3' (SEQ ID NO: 7).

[0024] Example 2: Extraction of nucleic acid from samples (1) The kit used was the RC323 viral nucleic acid extraction kit from Nanjing Novizan Pharmaceutical Co., Ltd. Add 20 μL of Proteinase K and 300 μL of sample to the RNase-free tube in sequence. If the sample volume is insufficient, use PBS or physiological saline to make up to 300 μL and 300 μL of Buffer VLPro working solution according to the sample volume. Vortex mix for 15-30 seconds, briefly centrifuge to collect the liquid on the tube cap and tube wall, and let stand at room temperature for 5 minutes.

[0025] (2) Add 200 μL of anhydrous ethanol, vortex mix for 15-30 seconds, briefly centrifuge to collect the liquid on the cap and tube wall. If there is precipitate of impurities at this time, it is normal and subsequent experiments can be carried out directly.

[0026] (3) Transfer all of the above mixture to FastPure DNA / RNA Columns, centrifuge at 12000 rpm (13800×g) for 1 minute, and discard the filtrate.

[0027] (4) Add 700 μL Buffer W1 to FastPure DNA / RNA Columns. Before use, please check whether anhydrous ethanol has been added. Centrifuge at 12000 rpm (13800×g) for 30 seconds and discard the filtrate.

[0028] (5) Add 700 μL of Buffer WW2 to FastPure DNA / RNA Columns, centrifuge at 12000 rpm (13800×g) for 30 seconds, and discard the filtrate.

[0029] (6) Centrifuge the empty column at 12000 rpm (13800×g) for 2 minutes.

[0030] (7) Transfer FastPure DNA / RNA Columns to a new RNase-free Collection Tube 1.5 mL, add 30-50 μL of RNase-free ddH2O to the center of the membrane, let stand at room temperature for 1 minute, and centrifuge at 12000 rpm (13800×g) for 1 minute.

[0031] (8) Discard FastPure DNA / RNA Columns. The extracted DNA / RNA can be used directly for subsequent detection. For short-term storage, store at -30℃ to -15℃. For long-term storage, store at -85℃ to -65℃.

[0032] Example 3 RNA Reverse Transcription For RNA samples, reverse transcription was performed according to the instructions of the RevertAid First Strand cDNA Synthesis Kit. The reaction system is shown in Table 1.

[0033] Table 1 Reverse transcription system

[0034] The reaction conditions were: complete chain deactivation at 25°C for 20 minutes, annealing at 42°C for 45 minutes, and extended inactivation at 72°C for 5 minutes.

[0035] Example 4 Preparation of positive plasmid standards This embodiment provides a method for preparing positive plasmid standards for constructing dual qPCR standard curves.

[0036] The PBFDV Rep gene fragment and APV VP1 gene fragment in positive samples were amplified using long-fragment amplification primer pairs BFDV-F / R (SEQ ID NO: 1 / 2) and APV-VP-F / R (SEQ ID NO: 6 / 7), respectively. The PCR products were verified by agarose gel electrophoresis, as shown below. Figure 1 As shown, the PCR product containing the target fragment was cloned into the pEASY®-Blunt Zero cloning kit vector, transformed into competent cells, and the positive plasmid was extracted. Verification was performed using Sanger sequencing. The correctly sequenced recombinant plasmids were named pEASY-BFDV and pEASY-APV, respectively. Figure 1 In the study, M represents the DL2000 marker; lanes 1 and 3 are negative controls; and lanes 2 and 4 are positive controls.

[0037] Plasmid concentration was measured using Nanodrop and converted to copies / μL. The initial plasmid concentration was pEASY-BFDV 5.3 × 10⁻⁶. 10 copies / μL, pEASY-APV 4.5×10 10 copies / μL. Adjust both concentrations to 2×10⁻⁶. 9 Copies / μL were prepared as a stock solution. The stock solution was serially diluted 10-fold to serve as a standard for subsequent construction of standard curves and sensitivity testing.

[0038] Example 5: Validation of Single qPCR Primer-Probe Combinations To ensure the accuracy and reliability of primers and probes, each combination was first incorporated into the qPCR reaction system, and the corresponding plasmid containing the target gene was used as a template for verification.

[0039] The APV singlet qPCR reaction system is shown in Table 2.

[0040] Table 2 APV singlet qPCR reaction system Reagent volume (μL)

[0041] The PBFDV singlet qPCR reaction system is shown in Table 3.

[0042] Table 3 PBFDV singlet qPCR reaction system

[0043] The reaction program was as follows: 95°C pre-denaturation for 30 seconds; 40 cycles of 95°C denaturation for 5 seconds, 60°C annealing and extension for 34 seconds.

[0044] Only when specific amplification curves and Ct values ​​can be detected when using corresponding plasmids containing the target gene, proving the specificity and accuracy of the primers and probes, can subsequent double qPCR experiments be performed.

[0045] Example 6 Optimization of Dual qPCR Reaction Conditions To determine the optimal reaction conditions, two standard plasmids (10) were used. 8 Using copies / μL as templates, the annealing temperature, primer concentration, and probe concentration were optimized, and the optimal conditions were selected based on the cycle threshold (Ct) value and amplification efficiency.

[0046] (1) Optimization of primer concentration With other conditions remaining constant, experiments were conducted with primer final concentrations of 0.1 μM, 0.2 μM, 0.4 μM, 0.6 μM, and 0.8 μM, respectively. The results showed that the lowest Ct value and highest amplification efficiency were achieved when the APV primer concentration was 0.2 μM and the PBFDV primer concentration was 0.4 μM. Figure 2 As shown in A and D, where Figure 2 The blue, red, and green lines represent the Ct value of APV, the Ct value of PBFDV, and the amplification efficiency, respectively.

[0047] (2) Optimization of probe concentration With other conditions remaining constant, experiments were conducted with probe final concentrations of 0.2 μM, 0.4 μM, 0.6 μM, 0.8 μM, and 1.0 μM, respectively. The results showed that when both the APV and PBFDV probe concentrations were 0.8 μM, the Ct value was the lowest and the amplification efficiency was the highest. Figure 2 As shown in B and E.

[0048] (3) Optimization of annealing temperature With other conditions remaining constant, experiments were conducted at annealing temperatures of 56℃, 58℃, 60℃, and 62℃. The results showed that the lowest Ct value and highest amplification efficiency were observed at an annealing temperature of 58℃. Figure 2 As shown in C and F.

[0049] (4) Optimization of the number of loops Experiments were conducted with 30, 35, 40, 45, and 50 cycles respectively. The results showed that 30 and 35 cycles led to insufficient amplification, while 45 and 50 cycles led to increased background signal and nonspecific amplification. Therefore, 40 cycles was selected as the optimal number of cycles.

[0050] Based on the above optimization results, the optimal reaction system and reaction procedure were determined.

[0051] Example 7: Dual qPCR Detection Method This embodiment provides a specific method for dual real-time PCR detection using the primer and probe combination described in Example 1.

[0052] The dual qPCR reaction system is shown in Table 4.

[0053] Table 4 Dual qPCR reaction system

[0054] The double qPCR reaction program was as follows: 95℃ pre-denaturation for 30 seconds; 40 cycles of 95℃ denaturation for 5 seconds, 58℃ annealing and extension for 34 seconds.

[0055] Amplification was performed on an ABI QuantStudio 5 real-time quantitative PCR instrument. Fluorescence signals were collected in the FAM channel (for detecting APV) and the VIC channel (for detecting PBFDV). The presence of PBFDV and / or APV in the sample was determined based on the amplification curve and Ct value: if the sample showed a typical S-shaped amplification curve in the FAM channel and the Ct value was ≤38, it was considered APV positive; if the sample showed a typical S-shaped amplification curve in the VIC channel and the Ct value was ≤38, it was considered PBFDV positive; if amplification curves were observed in both channels, it was considered a mixed infection.

[0056] Example 8: Establishment of a Dual qPCR Standard Curve The pEASY-APV and pEASY-BFDV positive plasmid standards prepared in Example 4 were mixed and serially diluted 10-fold, with concentrations starting from 1×10⁻⁶. 6 The mixture of 1×10³ copies / μL was used as a template, and double qPCR amplification was performed according to the reaction system and reaction procedure of Example 7 to establish a standard curve.

[0057] The results are as follows Figure 3 As shown, the standard curve equation for APV is: y = -3.456x + 40.954 (R² = 0.997, amplification efficiency Eff% = 98.5%); the standard curve equation for PBFDV is: y = -4.115x + 46.645 (R² = 0.995, amplification efficiency Eff% = 95.8%). The R² values ​​of both viral standard curves are greater than 0.99, and the amplification efficiencies are both within the ideal range of 90%-110%, indicating that these standard curves have good linearity and amplification efficiency, and can be used for subsequent absolute quantitative detection of samples.

[0058] Example 9: Dual qPCR Specificity Assay To verify the specificity of the dual qPCR detection method described in this invention for PBFDV and APV, nucleic acids from various viruses and pathogens were used as templates for specificity verification.

[0059] DNA templates for APV, PBFDV, MDV, ALV, IBDV, NDV, CIAV, and Chlamydia psittaci were used respectively, and double qPCR amplification was performed according to the reaction system and procedure of Example 7, with a template-free negative control set up.

[0060] The results are as follows Figure 4 As shown, specific amplification curves were detected only in the DNA templates of APV and PBFDV. APV showed a typical S-shaped amplification curve in the FAM channel, and PBFDV showed a typical S-shaped amplification curve in the VIC channel. No amplification signals were detected in other non-target pathogens (MDV, ALV, IBDV, NDV, CIAV, and Chlamydia psittaci). The negative control (no template control) was also negative. The above results indicate that the dual qPCR detection method described in this invention has high specificity and can accurately distinguish between target viruses and non-target pathogens.

[0061] Example 10 Dual qPCR Sensitivity Assay To determine the limit of detection (LoD) of the dual qPCR detection system described in this invention, a gradient dilution method was used to detect and verify the positive plasmids.

[0062] pEASY-APV and pEASY-BFDV positive plasmid standards were serially diluted 10-fold (10... 6 Up to 10 0 (copies / μL), each dilution was vortexed to mix, 25 replicates were set for each concentration gradient, and ddH2O was set as a blank control. Dual qPCR amplification was performed according to the reaction system and reaction procedure of Example 7, and the Ct value and detection rate of each concentration were recorded.

[0063] The average Ct values ​​and detection rates of plasmid standards with different gradients are shown in Table 5.

[0064] Table 5. Average Ct values ​​and detection rates of plasmid standards with different gradients

[0065] Amplification curves as follows Figure 5 As shown, the detection limit was calculated using PROBIT regression analysis, and the results are as follows. Figure 6 As shown, the detection limit (LoD) for APV was 7.264 copies (95% confidence interval: 5.901-17.676 copies), and the detection limit (LoD) for PBFDV was 11.935 copies (95% confidence interval: 10.229-14.992 copies). These results indicate that the dual qPCR detection method described in this invention has high sensitivity, with the detection limit for both viruses reaching the single-copy level, and can effectively detect low-viral-load samples.

[0066] Example 11 Dual qPCR Repeatability Experiment To evaluate the repeatability and stability of the dual qPCR detection method for detecting PBFDV and APV described in this invention, intra-group and inter-group replication experiments were performed.

[0067] Within-group replication: with high (1×10) 6 copies / μL), medium (1×10) 4 Using positive plasmid standards at three concentrations (copies / μL) and high (1×10² copies / μL) as templates, three replicates were set for each concentration within the same batch. The detection was performed according to the reaction system and reaction procedure of Example 7, and the mean, standard deviation (SD), and coefficient of variation (CV) of Ct values ​​for each concentration were calculated.

[0068] Intergroup replication experiment: Using the same three concentrations of positive plasmid standards as templates, the test was repeated 3 times in different batches, and the mean, standard deviation (SD) and coefficient of variation (CV) of Ct values ​​for each concentration were calculated.

[0069] The results of the repeatability experiment are shown in Table 6.

[0070] Table 6 Results of within-group and between-group replicate experiments

[0071] The results showed that the coefficients of variation (CV) of the within-group and between-group Ct values ​​of APV and PBFDV were all less than 2% at high, medium and low concentration levels, confirming that the dual qPCR detection method described in this invention has excellent repeatability and stability, and the detection results are reliable.

[0072] Example 12 Dual qPCR Clinical Sample Detection To verify the clinical applicability of the dual qPCR detection method described in this invention, 702 clinical samples were tested using the established dual qPCR method, and a parallel comparison was made with a published single qPCR method.

[0073] (1) Sample collection and processing A total of 702 clinical samples were collected in this study, including 56 liver tissue samples, 158 cloacal swabs and 488 feather swabs. These samples were obtained from parrot farms and individual breeders in Central China, North China and East China. All samples were transported on ice immediately after collection and stored at -80°C for later use. Total nucleic acid was extracted from each sample according to the method in Example 2.

[0074] (2) Clinical sample testing 702 clinical samples were tested in parallel using the dual qPCR method described in this invention (Example 7) and the published single qPCR method. For dual qPCR testing, each sample was tested in two replicate wells, and the average Ct value was used for determination. The determination criteria were as follows: APV positive was indicated by a typical S-shaped amplification curve in the FAM channel and a Ct value ≤ 38; PBFDV positive was indicated by a typical S-shaped amplification curve in the VIC channel and a Ct value ≤ 38; and mixed infection was indicated by positive results in both channels.

[0075] The results of clinical sample testing are shown in Table 7.

[0076] Table 7 Clinical Sample Test Results

[0077] (3) Results Analysis Overall, among the 702 clinical samples, 231 were positive for PBFDV (32.91%), 32 were positive for APV (4.56%), and 18 were double-infected (2.56%). Compared with the single qPCR method, the double qPCR method described in this invention showed completely consistent detection results for all positive samples, with a concordance rate of 100%.

[0078] Analysis by sample type: The highest positivity rate of PBFDV was found in liver tissue (37.50%, 21 / 56), followed by cloacal swabs (34.81%, 55 / 158) and feather swabs (28.07%, 137 / 488). The highest positivity rate of APV was found in liver tissue (14.29%, 8 / 56), significantly higher than that of cloacal swabs (3.16%, 5 / 158) and feather swabs (3.89%, 19 / 488). Dual infection was most frequently detected in feather swabs (2.46%, 12 / 488), suggesting that feather swabs, as a non-invasive sample, have important value in monitoring mixed infections.

[0079] By region (as shown in Table 8), the PBFDV positivity rate in Central China was 36.21% (105 / 290), and the APV positivity rate was 5.17% (15 / 290); in North China, the PBFDV positivity rate was 31.52% (75 / 238), and the APV positivity rate was 4.20% (10 / 238); and in East China, the PBFDV positivity rate was 29.31% (51 / 174), and the APV positivity rate was 4.02% (7 / 174). There was no statistically significant difference in positivity rates among the regions (P>0.05), indicating that both viruses are prevalent in the above-mentioned areas.

[0080] Table 8 Comparison of PBFDV and APV positivity rates in different regions

[0081] The above clinical sample test results show that the dual qPCR detection method described in this invention has good clinical applicability, the detection results are completely consistent with the single qPCR method, and it has the advantages of high throughput and low cost. It is suitable for large-scale epidemiological monitoring of PBFDV and APV and rapid detection of clinical samples in parrot farms.

[0082] Example 13 Application of feather swab samples in mixed infection surveillance This embodiment further verifies the application value of feather swabs as non-invasive samples in the monitoring of mixed PBFDV and APV infections.

[0083] The dual qPCR method described in Example 7 was used to test 488 parrot feather swab samples. The specific procedure was as follows: 3-5 feathers were taken from the chest or back of a parrot using sterile forceps. The base (follicle end) of each feather was cut into a 1.5 mL centrifuge tube, 500 μL of PBS buffer was added, the tube was vortexed for 30 seconds, and after brief centrifugation, the supernatant was collected. Total nucleic acid was extracted using the method in Example 2, and then dual qPCR detection was performed using the method in Example 7.

[0084] The test results showed that 137 out of 488 feather swab samples (28.07%) were positive for PBFDV. Nineteen samples (3.89%) were APV positive, and 12 samples (2.46%) were double-infected. The detection rate of double infection in feather swabs was the highest among the three types of samples (2.46%, 12 / 488), suggesting that feather swabs, as a non-invasive sample, have important value in the monitoring of mixed infections, and are especially suitable for non-invasive sampling monitoring of rare parrot species.

[0085] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, are covered within the scope of protection of the present invention.

Claims

1. A dual-fluorescent quantitative PCR primer and probe combination for simultaneously detecting Psittacine beak and feather disease virus and Avian polyomavirus, characterized in that, include: A first primer pair and a first probe for detecting parrot beak feather disease virus, wherein the first primer pair comprises a first forward primer and a first reverse primer, the nucleotide sequence of the first forward primer is shown in SEQ ID NO:3, the nucleotide sequence of the first reverse primer is shown in SEQ ID NO:4, and the nucleotide sequence of the first probe is shown in SEQ ID NO:

5. The second primer pair and the second probe are for detecting avian polyomavirus. The second primer pair consists of the first forward primer and the second reverse primer. The nucleotide sequence of the second forward primer is shown in SEQ ID NO:8, the nucleotide sequence of the second reverse primer is shown in SEQ ID NO:9, and the nucleotide sequence of the second probe is shown in SEQ ID NO:

10.

2. The primer and probe combination according to claim 1, characterized in that, The first probe has a VIC fluorescent group labeled at its 5' end and a BHQ1 quencher group labeled at its 3' end; the second probe has a FAM fluorescent group labeled at its 5' end and a BHQ1 quencher group labeled at its 3' end.

3. A dual real-time PCR kit for simultaneously detecting psittacosis beak feather virus and avian polyomavirus, characterized in that, It includes the primer and probe combination as described in claim 1 or 2.

4. The reagent kit according to claim 3, characterized in that, It also includes a positive plasmid standard containing the Rep gene fragment of psittacosis virus and the VP1 gene fragment of avian polyomavirus. The positive plasmid standard was cloned and constructed by amplifying the Rep gene fragment of psittacosis virus and the VP1 gene fragment of avian polyomavirus using the first construction primer pair SEQ ID NO:1 and NO:2 and the second construction primer pair SEQ ID NO:6 and NO:7, respectively.

5. A dual-fluorescence quantitative PCR method for simultaneous detection of psittactic beak feather disease virus and avian polyomavirus, characterized in that, the primer and probe combination described in claim 1 or 2 is used for dual-fluorescence quantitative PCR amplification. Includes the following steps: (1) Extract total nucleic acid from the sample to be tested, and use the total nucleic acid as a template. (2) Collect fluorescence signals and determine the presence of parrot beak feather disease virus and / or avian polyomavirus in the sample based on the amplification curve and Ct value.

6. The method according to claim 5, characterized in that, The reaction system for the dual quantitative PCR is as follows: 10 μL of 2×TaqMan probe-based quantitative PCR premix, 0.2-0.4 μL each of the second forward primer and the second reverse primer, 0.2-0.4 μL each of the first forward primer and the first reverse primer, 0.6-0.8 μL each of the second probe and the first probe, 2 μL of DNA template, and the remainder is nuclease-free water, for a total system volume of 20 μL.

7. The method according to claim 5, characterized in that, The reaction procedure for the dual real-time PCR is as follows: 95°C pre-denaturation for 30 seconds; 40 cycles of 95°C denaturation for 5 seconds, 58°C annealing and extension for 34 seconds.

8. The method according to claim 5, characterized in that, The samples to be tested were selected from liver tissue, cloacal swabs, or feather swabs.

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